Abstract/Summary

High-throughput pyrosequencing and quantitative PCR (Q-PCR) analysis offer greatly improved
accuracy and depth of characterisation of lower respiratory infections. However, such approaches
suffer from an inability to distinguish between DNA derived from viable and non-viable bacteria. This
discrimination represents an important step in characterising microbial communities, particularly in
contexts with poor clearance of material or high antimicrobial stress, as non-viable bacteria and
extracellular DNA can contribute significantly to analyses. Pre-treatment of samples with propidium
monoazide (PMA) is an effective approach to non-viable cell exclusion (NVCE). However, the impact
of NVCE on microbial community characteristics (abundance, diversity, composition and structure)
is not known. Here, adult cystic fibrosis (CF) sputum samples were used as a paradigm. The effects
of PMA treatment on CF sputum bacterial community characteristics, as analysed by pyrosequencing
and enumeration by species-specific (Pseudomonas aeruginosa) and total bacterial Q-PCR,
were assessed. At the local community level, abundances of both total bacteria and of P. aeruginosa
were significantly lower in PMA-treated sample portions. Meta-analysis indicated no overall
significant differences in diversity; however, PMA treatment resulted in a significant alteration in
local community membership in all cases. In contrast, at the metacommunity level, PMA treatment
resulted in an increase in community evenness, driven by an increase in diversity, predominately
representing rare community members. Importantly, PMA treatment facilitated the detection of both
recognised and emerging CF pathogens, significantly influencing ‘core’ and ‘satellite’ taxa group
membership. Our findings suggest failure to implement NVCE may result in skewed bacterial
community analyses.

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